Upregulation of adenosine A2A receptor and downregulation of GLT1 is associated with neuronal cell death in Rasmussen's encephalitis

Abstract

Rasmussen encephalitis (RE) is a severe pediatric inflammatory brain disease characterized by unilateral inflammation and atrophy of the cerebral cortex, drug-resistant focal epilepsy and progressive neurological and cognitive deterioration. The etiology and pathogenesis of RE remain unclear. Our previous results demonstrated that the adenosine A1 receptor (A1R) and the major adenosine-removing enzyme adenosine kinase play an important role in the etiology of RE. Because the downstream pathways of inhibitory A1R signaling are modulated by stimulatory A2AR signaling, which by itself controls neuro-inflammation, glial activation and glial glutamate homeostasis through interaction with glutamate transporter GLT-1, we hypothesized that maladaptive changes in adenosine A2A receptor (A2AR) expression are associated with RE. We used immunohistochemistry and Western blot analysis to examine the expression of A2ARs, glutamate transporter-I (GLT-1) and the apoptotic marker Bcl-2 in surgically resected cortical specimens from RE patients (n = 18) in comparison with control cortical tissue. In lesions of the RE specimen we found upregulation of A2ARs, downregulation of GLT-1 and increased apoptosis of both neurons and astroglia. Double staining revealed colocalization of A2ARs and Bcl-2 in RE lesions. These results suggest that maladaptive changes in A2AR expression are associated with a decrease in GLT-I expression as a possible precipitator for apoptotic cell loss in RE. Because A2AR antagonists are already under clinical evaluation for Parkinson's disease, the A2AR might likewise be a tractable target for the treatment of RE.

Keywords: Rasmussen encephalitis; adenosine A2A receptor; apoptosis; epilepsy; glutamate transporter-I.

Figure 1

Characteristic neuropathologic findings in brain tissue from RE patients. A. Microglial infiltrates and diffuse microglial activation in the cerebral cortex (arrow, H&E stain). B. Perivascular lymphocytic cuffing in the cerebral cortex (arrow, H&E staining). C. Parenchymal lymphocytic infiltrates in the cerebral cortex (arrows). D–F. Co‐localization of CD3 and CD8 immunoreactivity in lymphocytic infiltrates in the cerebral cortex (D, CD3 immunostaining; E, CD8 immunostaining; F, co‐localization of CD3 and CD8). G. Neuronal loss (arrows, NeuNimmunostaining). H. Ectopic neurons in the white matter (arrow, NeuNimmunostaining). I. Enlarged dysmorphic neurons in the white matter (arrow, inset, NeuN immunostaining). J. Radial microcolumns in the cortex (arrow, NeuN immunostaining). K–L. Reactive astrogliosis (GFAP immunostaining) in gray matter (K) and white matter (L). Scale bar: (A–C, G–L) 25 μm; (D–F) 12.5 μm.

Figure 2

Expression of A2ARs within the lesions of RE. A,B,E,F. Weak immunostaining for A2ARs in control cortical gray matter (A,E) and white matter (B,F). Negative or weak expression in neurons in cortical gray matter (E, arrow, inset) and sparse expression on glial cells in control white matter (F, arrow, inset). C,G. Marked ectopic cytoplasmic expressionof A2ARs in the remaining neuronal cells (G, arrow, inset)in gray matter within the lesion of RE. D,H. Marked ectopic cytoplasmic expression of A2ARs in reactive astrocytes (H, arrow, inset) in white matter within the lesion of RE. C and D shows absence of neuronal or astroglial immunoreactivity after pre‐absorption respectively. I,J. Marked expression of A2ARs in perivascular reactive astrocytes in white matter (I,J), and endothelial cells within the lesions of the cortex (J, arrow). In addition, marked cytoplasmic staining of A2ARs in ectopic neurons (enlarged dysmorphic neurons) was detected in the white matter (I, arrow). Scale bars: (A–D) 50 μm; (E–H) 25 μm, (I,J) 6.25 μm.

Figure 3

In situ hybridization to assess A2AR mRNA within the lesions of RE. A: Marked expression of A2AR mRNA within the lesions of RE; B: Very weak or negative expression of A2AR mRNA in control specimen. Scale bars: 12.5 μm.

Figure 4

A2AR expression in neurons, astrocytes and microglia in RE (case 8). A‐D. Co‐locolization of NeuN (A, green) and A2ARs (B, red) demonstrated ectopic cytoplasmic expression of A2ARs in the remaining neurons within the lesion area (D, arrow). E–H. Co‐localization of GFAP (E, green) and A2ARs (F, red) indicated ectopic cytoplasmic expression in reactive astrocytes within the lesion area (H, arrows). I–L. Co‐localization of Iba‐1 (I, green) and A2ARs (J, red) within the lesion area (L, arrows). Merged images (D,H,L, yellow) confirm localization of the A2AR in neurons, astrocytes and microglia (C,J,K). Representative micrographs of nuclear staining with DAPI. Scale bars: 15 μm.

Figure 5

Western blot analysis of A2ARs in RE and control specimens. A. Representative immunoblots of A2ARs in total homogenates of RE and control specimens. β‐actinimmunoreactivity was used to normalize for equal protein loading. B. Quantitative analysis of A2AR expression levels. Data are mean ± SEM (n = 5 per group). *P < 0.05 vs control.

Figure 6

Downregulation of the GLT‐1within the Lesions of RE. A,E. In control gray matter, marked staining and patchy distribution of GLT‐1 immunoreactivity detected in glial cell processes (E, inset, arrows). B,F. Sparse GLT‐1 expression on astrocytes in control white matter (F, inset, arrows). In specimens of RE, no detectable or only sparse GLT‐1 expression was detected on astrocytes in gray matter of RE (C,G, inset arrows) and white matter (D,H, inset arrows). Scale bars: A–D, 50 µm; E‐H, 25 µm. (I) Scanned immunodensities of GLT‐1 expression show asignificant decrease (P < 0.01) in GLT‐1 expression within the RE (n = 18) grey matter compared with controls (n = 6).

Figure 7

Downregulation of Bcl‐2 within RE lesions. A,B,E,F. Bcl‐2 immunoreactivity in neurons of control grey matter (A,E, inset, arrows) and in the astrocytes within control white matter (B,F, inset, arrows). C and G. No detectable (G, inset, arrows) or sparse weak neuronal expression (G, inset, arrowheads) of Bcl‐2 in gray matter of RE. D and H. no detectable (H, inset, arrows) or sparse weak astrocytic expression (H, inset, arrowheads) in white matter of RE. Scale bars: A–D, 50 µm; E–H, 25 µm. (I) Immunohistochemistry analysis demonstrated a significant decrease (P < 0.05) in the density of Bcl‐2 immunoreactivity within the RE (n = 18) gray matter compared with controls (n = 6).

Figure 8

Co‐localization of A2ARs and TUNEL labeling in RE lesions (case 8). A–D. Co‐localization of A2AR (red) (A) with TUNEL (green) (B) (D, arrows) in the gray matter of RE. E–H. Co‐localization of A2ARs (red) (E) with TUNEL (green) (F) (H, arrows) in the white matter of RE. All slices were conterstained with the nuclear marker DAPI. Scale bars: 15 μm.

Figure 9

Expression of CD73 within the lesions of RE. A,E. Marked ectopic cytoplasmic expression of CD73 in the remaining neuronal cells (E, arrow, inset) in gray matter within the lesion of RE. B and F. Marked cytoplasmic expression of CD73 in reactive astrocytes (F, arrow, inset) in white matter within the lesion of RE. C,D,G,H. Very weak or negative immunostaining of CD73 in control cortical gray matter (C,G) and white matter (D,H). I. Immunodensities of CD73 expression show a significant decrease (P < 0.05) in CD73 expression within the RE (n = 18) compared with controls (n = 6). Scale bars: (A–D) 50 μm; (E‐H) 25 μm.